香草酸与多酚氧化酶的相互作用机制及新型指纹谱构建  

Interaction Mechanism and Three-dimensional Fingerprint of Vanillic Acid Binding with Polyphenol Oxidase

作　　者：郭明[1] 詹敏忠[1] 胡智燕[1] 成纪予[2] 

机构地区：[1]浙江农林大学理学院,浙江临安311300 [2]浙江农林大学农业与食品科学学院,浙江临安311300

出　　处：《中国生物化学与分子生物学报》2014年第12期1236-1243,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.31301580);浙江省自然科学基金项目(No.LY13C200012)资助~~

摘　　要：生物大分子与小分子之间的相互作用机制研究是当今各个学科领域的前沿和热点,不仅有利于进一步认识大分子的结构和功能,还能进一步获得检测生物大分子或小分子的新途径.本研究将中药材特征指纹图谱应用于植物多酚氧化酶(polyphenol oxidase,PPO)与植物体内活性成分香草酸(vanillic acid,VA)的具体相互作用机制的研究,采用光谱实验法结合分子模拟技术,分析VA与PPO的相互作用机制,并构建其三维相互作用指纹谱.光谱实验结果显示,VA增强了PPO的荧光强度.维持VA-PPO体系的相互作用力主要为疏水作用,VA与PPO的结合距离r值为2.48 nm,发生了非辐射能量转移.由光谱实验数据构建的λ-UV-F新型指纹图谱,系统地反映了活性分子VA与PPO之间相互作用特征.分子模拟结果精确显示了VA与PPO的结合位域与结合作用力,表明维持VA与PPO的相互作用力主要为疏水作用和氢键(位于氨基酸残基Met258,His88,His109,His240,His244和His274位).计算机模拟与光谱学实验结果一致,并成功构建了VA-PPO相互作用特征关系的新型指纹图谱.Research about the interaction mechanism between biomacromolecules and small molecules has become a frontier and hot topic in the field of various subjects, which is not only beneficial to further understand the structure and function of biomacromolecules, but also can further obtain new ways of detecting biomacromolecules and small molecules. In the present domestic and overseas investigation, the research of biomacromolecules interaction with small molecules are more concentrated on binding reaction between proteins and drug molecules, or focuses on the reaction between animals protease and drug molecu}es. Moreover, the study about the interaction between plant enzymes and active plant component is very rare reported. The Chinese herbal medicine fingerprints were used to analyze the interaction between vanillic acid （VA） and polyphenol oxidase （PPO） and the interaction mechanisms were explored by using fluorescence spectroscopy/UV-visible absorbance spectroscopy in combination with molecular modeling studies. The three-dimensional fingerprint of the VA-PPO system was built. The fluorescence spectroscopy indicated that the interaction between PPO and VA resulted in fluorescence enhancement. The thermodynamic parameters suggested that the hydrophobic interaction predominantly stabilized the complex. The values of r （2.48 nm） was lower than 7 nm after interaction between protein PPO and the VA molecule, which indicated the efficiency of energy transfer according to Forster theory. The VA-PPO λ-UV-F fingerprint was built by using the spectroscopic data, which revealed the interaction of the active molecule VA binding with PPO. Molecular model revealed one major hydrophobic interaction and six hydrogen bond interactions between VA and the residues Met258, His88, His109, His240, His244 and His274 of PPO. The computational simulations showed consistent results with spectral experiment. This work also built a three-dimensional fingerprint of the VA-PPO system.

关 键 词：香草酸 多酚氧化酶 荧光加强 分子模拟 

分 类 号：Q5[生物学—生物化学]